RESEARCH ARTICLE

Delayed-Type Hypersensitivity to Metals of

Environmental Burden in Patients with

Takotsubo Syndrome – Is There a Clinical

Relevance?

Jan Manousek1, Vera Stejskal2,3, Petr Kubena1, Jiri Jarkovsky4, Petr Nemec5,6,

Petr Lokaj1,6, Ludmila Dostalova1, Andrea Zadakova1, Marie Pavlusova1,6,

Klara Benesova4, Petr Kala1,6, Roman Miklik1, Jindrich Spinar1,6, Jiri Parenica1,6*

1 Department of Internal Medicine and Cardiology, University Hospital Brno, Brno, Czech Republic,

2 Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm,

Sweden, 3 Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science,

Masaryk University, Brno, Czech Republic, 4 Institute of Biostatistics and Analyses, Faculty of Medicine,

Masaryk University, Brno, Czech Republic, 5 Centre of Cardiovascular Surgery and Transplantations, Brno,

Czech Republic, 6 Faculty of Medicine, Masaryk University, Brno, Czech Republic

* jiri.parenica@atlas.cz

Abstract

Objective

Takotsubo syndrome (TS) is a heart condition characterised by a sudden transient left ventricu-

lar dysfunction; its pathophysiology is probably associated with elevated levels of catechol-

amines but the exact mechanism is not known as yet. Literature and clinical experience

suggest that TS affects persons with various comorbidities. This pilot work aims to evaluate the

frequency of comorbidities with potential pathological immune reactivity, and to evaluate the

potential association between TS and hypersensitivity to metals assessed by LTT-MELISA®.

Methodology, Results

A total of 24 patients (23 women, 1 man) with a history of TS attack and 27 healthy controls

were evaluated. Hypersensitivity was evaluated by a lymphocyte transformation test (LTT-

MELISA®); a questionnaire of environmental burden was used to select evaluated metals. A

total of 19 patients (79%) had at least one condition that might potentially be associated with

pathological immune reactivity (autoimmune thyroid disease, drug allergy, bronchial asthma,

cancer, contact dermatitis, rheumatoid arthritis). Hypersensitivity to metals was identified sig-

nificantly more frequently in TS patients than in healthy controls (positive reaction to at least

one metal was identified in 95.8% of TS patients and in 59.3% of controls; p = 0.003); the dif-

ference was statistically significant for mercury (45.8% and 14.8%, respectively; p = 0.029).

Conclusion

Our work shows that conditions with pathological immune reactivity occur frequently in TS

patients, and our data suggest a possible association between TS and hypersensitivity to

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 1 / 12

a11111

OPENACCESS

Citation: Manousek J, Stejskal V, Kubena P,

Jarkovsky J, Nemec P, Lokaj P, et al. (2016)

Delayed-Type Hypersensitivity to Metals of

Environmental Burden in Patients with Takotsubo

Syndrome – Is There a Clinical Relevance? PLoS

ONE 11(11): e0164786. doi:10.1371/journal.

pone.0164786

Editor: Giuseppe Andò, University of Messina,

ITALY

Received: March 19, 2016

Accepted: October 1, 2016

Published: November 8, 2016

Copyright: © 2016 Manousek et al. This is an open

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the paper and its Supporting Information

files.

Funding: This work was supported by Ministry of

Health of the Czech Republic – conceptual

development of research organisation (FNBr,

65269705), project MUNI/A/1362/2015 of the

Masaryk University (Brno, Czech Republic) and by

a grant of the Czech Society of Cardiology.

metals (mercury in particular) evaluated by LTT-MELISA®. We also suggest that apart

from the triggering stress factor, potential existence of other serious conditions should be

considered when taking medical history of TS patients.

Introduction

Takotsubo syndrome (TS) is a relatively rare heart condition that was first described by Satoet al. in 1990 [1]. At the time of acute attack, TS is characterised by transient left ventriculardysfunction, which most frequently affects apical segments of the left ventricle, and which can-not be explained by coronary angiography (showing a normal finding) [2]. Differential diagno-sis must primarily exclude acute myocardial infarction and acute myocarditis [3].Postmenopausal women are particularly affected, and mental stress (28%) or physical stress(36%) precedes an acute manifestation of TS [4]. Even after 25 years since the first written ref-erence of TS, the actual cause of the condition remains unknown. Various aetiological andpathological concepts have been suggested, such as catecholamine cardiotoxicity in case ofhigh plasma levels of these substances; this hypothesis has been supported by most clinical andexperimental evidence. Changes in the microcirculation with endothelial dysfunction and cor-onary artery spasms, alterations in calcium-regulatory proteins, or hormonal effects have alsobeen suggested [2,5]. It has been recently suggested to distinguish clinically between primaryTS (where acute cardiac symptoms are the primary reason for seeking medical care) and sec-ondary TS (where an acute attack develops in patients originally hospitalised for other reasons)[6]. At the present time, it is obvious that TS patients tend to have other comorbidities, oftenchronic ones [7], such as malignant tumours treated with chemotherapy [8], serious diseases ofthe gastrointestinal tract [9], psychiatric disorders [7], or neurological disorders [10–12]. Lungconditions are relatively common, such as the chronic obstructive pulmonary disease (COPD)or bronchial asthma, which are reported in 22–44% of patients [7,13]. Acute attacks of TS wererepeatedly described after the administration of some drugs [14] (e.g. allopurinol [15], 5-fluo-rouracil [8,16], oxaliplatin [17]), or early after pacemaker implantation [18].

The everyday clinical practice suggests that not all (even serious) cases of stress burden orphysical burden lead to the development of an acute attack of TS. Based on our clinical experi-ence and published results, we were intrigued by the fact that many TS patients suffer from var-ious chronic conditions, often associated with hypersensitivity reactions. On the other hand,hypersensitivity to metals was demonstrated in many patients with autoimmune disorders,including autoimmune thyroiditis [19], contact dermatitis [20] or connective tissue disease[21]. Hypersensitivity to metals is a type IV hypersensitivity (delayed-type hypersensitivity,DTH), which is mediated by specific T-lymphocytes. Under physiological conditions, this reac-tion is directed against intracellular pathogens (viruses, Chlamydia, Borrelia) and cancer cells.Most frequently, it is caused by a chronic exposure to low levels of antigen–hapten. Hypersen-sitivity can be evaluated by the so-called LTT-MELISA1 (Memory Lymphocyte Immunosti-mulation Assay). It is a modified lymphocyte transformation test (LTT), which is based on theprinciple of antigen/allergen-specific induction of cell division in lymphocytes following con-tact with their respective antigens. A positive reaction in the LTT indicates the presence of anti-gen-specific lymphocytes (memory cells) in the patient’s blood [22,23].

We formulated the hypothesis that hypersensitivity reaction might be associated with thedevelopment of TCM. Our work aimed to describe the occurrence of conditions potentiallyassociated with pathological immune reactivity in patients with a history of TS attack and,based on LTT-MELISA1, to evaluate the potential hypersensitivity of these patients to

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 2 / 12

Competing Interests: Vera Stejskal is owner of the

MELISA® trademark and receives royalties from

LTT-MELISA® tests. This does not alter our

adherence to PLOS ONE policies on sharing data

and materials.

antigens (metal pollutants) to which they are exposed according to the questionnaire of envi-ronmental burden. These results were then compared to results of the control group of individ-uals with no history of TS.

Materials and Methods

Ethical approval of the study protocol

Written informed consent forms were obtained from all subjects before their participation inthis project. The study protocol complied with the Declaration of Helsinki, and was approvedby the Ethics Committee of University Hospital Brno (Brno, Czech Republic).

Study population

The study involved patients hospitalised for an acute attack of TS from May 2006 to August2013 at the Department of Internal Medicine and Cardiology of the University Hospital Brno.A total of 24 patients (23 women, 1 man) were admitted to the coronary acute unit with sus-pected acute coronary syndrome, and subsequently were diagnosed with TS according to theMayo Clinic criteria [3]. ECG changes, hs-troponin T levels (Roche Diagnostics, Indianapolis,IN, USA, with the cut-off value at 14 ng/L), NT-proBNP (Roche Diagnostics, Indianapolis, IN,USA; with the cut-off value at 100 pg/mL), C-reactive protein (Roche, Basel, Switzerland, withthe cut-off value at 5 mg/L) (all biomarkers were measured at admission), coronary angiogra-phy finding, and left ventricular function (assessed by echocardiography and left ventriculogra-phy) were evaluated in all patients. The study involved only patients with a history of TS and adocumented recovery of left ventricular function. Some patients were also examined by mag-netic resonance imaging (MRI) in order to rule out ischaemic aetiology or acute myocarditis.The control group was composed of 27 healthy volunteers (20 women, 7 men) who had a simi-lar environmental burden with metals according to the questionnaire.

Evaluation of hypersensitivity reactions by LTT-MELISA®LTT-MELISA1 was performed as described previously [22]. Overall, 50 ml of venous bloodwere collected from each patient into tubes containing anti-coagulant and into 2 serum tubes.Blood samples were well isolated against cold and sent by overnight delivery to LaboratoireMGD in Switzerland for MELISA testing. Tested metals were selected according to informationon metal exposure provided in patients’ questionnaires. The following metals were tested:cobalt (Co), copper (Cu), inorganic mercury (Hg), nickel (Ni), silver (Ag), tin (Sn), beryllium(Be), cadmium (Cd), chromium (Cr), gold (Au), lead (Pb), molybdenum (Mo), palladium(Pd), manganese (Mn), and titanium (Ti).

Lymphocytes were isolated from the blood by density gradient centrifugation using Ficoll-PaqueTM media [19] and cultivated in RPMI 1640 Medium supplemented with 10% inactivatedhuman AB+ serum for 5 days at 37°C in a CO2 incubator. At least 2 different concentrations ofvarious metal salts were used for each patient. Three negative controls (lymphocytes cultivatedin the absence of metal salt) as well as a positive control (pokeweed mitogen, PWM) were alsoincluded. Lymphocyte proliferation in cultures was measured by the addition of isotope-labeled 3H thymidine for 4 hours, followed by harvesting of DNA on filters. The filters weresubsequently dried, scintillation fluid was added, and radioactivity retained on filters was mea-sured using a beta scintillation counter (Filtermat).

The stimulation index (SI) was used to evaluate the lymphocyte proliferative response. SIequals to counts per minute in metal-treated cultures divided by counts per minute in controlcultures cultivated without metal salt. SI� 3 was considered as positive response in our

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 3 / 12

evaluation. Generally value of SI between 2 and 3 is considered as a weakly positive response,while SI� 10 is considered as a strongly positive response.

The laboratory worker who evaluated LTT-MELISA1 did not know which samples weretaken from TCM patients or from healthy controls.

Environmental burden

The environmental burden was evaluated by a questionnaire which assessed the exposure of agiven person to metal pollutants that are mainly present in dental filling materials, prosthesesand implants, the person’s smoking burden, etc. The questionnaire was evaluated by a personwho did not know whether the questionnaire was completed by a TS patient or by a healthycontrol.

Statistical analysis

Stimulation indices were binarised according to cut-off SI� 3 (positive group), and the statisti-cal significance of relation of these positive groups to patient categories was tested using theFisher’s exact test. The analysis was calculated for each metal separately and for combined posi-tive groups of all metals together. The statistical significance of differences in continuous valuesof stimulation indices between categories of patients was tested using the Mann-Whitney test.

Results

The study cohort involved 24 patients with a history of TS; their basic characteristics areshown in Table 1. A total of 18 patients (75%) had apical variant of TS, while 6 patients (25%)had mid-ventricular variant of the condition. According to echocardiography, the median (5th,95th percentile) left ventricular ejection fraction (LVEF) in the acute phase was 42% (24%,57%); after the recovery of left ventricular function, LVEF was 65% (52%, 82%) (p< 0.001 forthe difference between LVEF in the acute phase and after the recovery of left ventricular func-tion, according to the Wilcoxon paired test). LVEF in healthy controls was 66% (56%, 73%)

Table 1. Basic characteristics of the TS group and the control group of healthy individuals.

TS group (N = 24) Controls (N = 27)

Sex–women 23 (96%) 20 (74%)

Median age (years; 5th-95th percentile) 65.5 (41.0–75.0) 58.0 (48.0–89.0)

Hypertension 14 (58%) 3 (11%)

Smoker or ex-smoker 10 (42%) 7 (26%)

Atrial fibrillation 6 (25%) 0

Corticosteroid therapy 5 (21%) 0

Depressive disorder 4 (17%) 1 (4%)

Diabetes mellitus 4 (17%) 0

Epilepsy 1 (4%) 0

Disorders potentially associated with immunopathological reaction:

-Autoimunne thyroid disease 7 (29%) 0

-Drug allergy 7 (29%) 3 (11%)

-Bronchial asthma 6 (25%) 0

-Cancer 4 (16%) 0

-Contact dermatitis 2 (8%) 7 (26%)

-Rheumatoid arthritis 1 (4%) 0

At least one immunopathological disorder 19 (79%) 8 (30%)

doi:10.1371/journal.pone.0164786.t001

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 4 / 12

(p = 0.719 for the comparison of LVEF in healthy controls and TS patients after the recovery ofleft ventricular function, according to the Mann–Whitney U test). At admission, TS patientshad high levels of NT-proBNP (median value of 525 pg/ml; 5th and 95th percentiles 150 pg/mland 15761 pg/ml, respectively) and relatively low levels of C-reactive protein (median value of5.98 mg/ml; 5th and 95th percentiles 0.57 mg/l and 107.33 mg/l, respectively). Mental stress,most frequently a death in the family, was the triggering factor in 6 women (25%).

Table 1 shows the most frequent comorbidities. Each patient in the TS group had at leastone of the mentioned comorbidities. Out of all comorbidities, we selected conditions thatmight potentially be associated with pathological immune reactivity (autoimmune thyroid dis-ease, drug allergy, bronchial asthma, cancer, contact dermatitis, rheumatoid arthritis). A totalof 19 patients (79%) in the TS group had at least one of these conditions.

Table 2 clearly demonstrates that environmental burden with metals is comparable betweenthe group of TS patients and the control group.

In the TS group, positive response (stimulation index SI� 3) to at least one of the evaluatedmetals was detected in a total of 23 patients (95.8%; 22 women, 1 man). The remaining onepatient (a woman) had only a weakly positive response for two metals (SI = 2.0 and 2.5). Elevenpatients (46%) had a positive response to one metal, eight patients (33%) to two metals, twopatients (8%) to three metals, and one patient (4%) to four metals; one patient (4%) had poly-valent allergy to six metals. Hypersensitivity reactions to nickel (11 patients, 46%) and mercury(10 patients, 42%) were most frequent, followed by hypersensitivity reactions to cobalt and sil-ver (4 patients, 17%), cadmium and lead (3 patients, 13%), copper (2 patients, 8%), tin andchromium (1 patient, 4%).

In the control group, positive response (stimulation index SI� 3) was detected in 16 per-sons (59.3%). Seven controls (26%) had a positive response to one metal, five controls (19%) totwo metals, and four controls (15%) to three metals. Hypersensitivity reaction to nickel (9 con-trols, 33%) was most frequent, followed by hypersensitivity reactions to mercury and gold (4controls, 15%), cadmium (3 controls, 11%), tin and titanium (2 controls, 7%), beryllium, chro-mium, cobalt and silver (1 control, 4%). Five persons from the control group did not have anyresponse to any evaluated metal, and 6 controls had only a weakly positive response (SI = 2.0–2.9) to 1–3 metals. In general, hypersensitivity reactions were milder and less frequent in thecontrol group when compared to the TS group.

Table 2. Overview of environmental burden with metals in the TS group and in the control group.

Metal of environmental burden TS group (N = 24) Controls (N = 27) Fisher exact test P-value

Ag 24 (100%) 27 (100%) 0.999

Al 12 (50%) 11 (41%) 0.579

Au 9 (37%) 11 (41%) 0.999

Cd 10 (42%) 11 (41%) 0.999

Co 7 (29%) 7 (26%) 0.999

Cu 24 (100%) 27 (100%) 0.999

Cr 17 (71%) 17 (63%) 0.766

Hg 24 (100%) 27 (100%) 0.999

Ni 17 (71%) 17 (63%) 0.766

Pb 10 (42%) 11 (41%) 0.999

Sn 24 (100%) 27 (100%) 0.999

Ti 24 (100%) 27 (100%) 0.999

Ag, Cu, Hg, Sn: amalgam alloy; Ni, Co, Cr: metal-bound ceramics, metal-bound bridge; Ni, Cr: stainless steel; Ti: titan alloy of dental implants,

pacemakers, implantable cardioverter defibrillators; Al: glass-ionomer cement; Al, Cd, Cr, Cu, Ni, Pb: cigarette smoke; Al, Ti: toothpastes, pills.

doi:10.1371/journal.pone.0164786.t002

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 5 / 12

Table 3 shows the numbers of positive LTT-MELISA1 tests (SI� 3) for evaluated metalsin TS patients and in healthy controls. TS patients had a statistically significantly higher fre-quency of hypersensitivity to mercury (45.8%) than the control group (14.8%; p = 0.029).

This statistically significant difference was evident even if we considered stimulationindex� 5 as the cut-off value: in this case, positive response was detected in 29.2% of TSpatients, and in 3.7% of healthy controls (p = 0.019).

When evaluating the number of patients who had a positive result of LTT-MELISA1 for atleast one metal, we found a statistically significant higher number of individuals with such reac-tivity in the group of TS patients than in the control group (95.8% and 59.3%, respectively;p = 0.003).

Discussion

Our work is the first one to point out two important issues in connection with TS: (1) a rela-tively frequent occurrence of conditions possibly linked to immunity, and (2) the possible linkbetween TS and delayed-type hypersensitivity (DTH) to metals of environmental burden, eval-uated by LTT-MELISA1. Apart from one patient with a weakly positive hypersensitivity reac-tion to two metals (stimulation index SI = 2.0 and 2.5), all other TS patients had a positiveresponse (SI� 3). This reactivity was statistically significantly more frequent in TS patientsthan in healthy controls. According to literature, the prevalence of hypersensitivity reactions tonickel (evaluated by patch tests) is high in the general population, estimated at 17–31% inwomen, and 3% in men; about 1–3% of the population are estimated to have hypersensitivityreactions to cobalt or chromium [24,25]. Evaluation of hypersensitivity reactions by LTT-ME-LISA1 detected a positive response to one metal in 43% of healthy controls, while 18% had a

Table 3. Comparison of a number of patients with positive stimulation index (SI� 3) for individual metals between TS group and healthy

controls.

TS group (N = 24) Controls (N = 27)

SI� 3 SI� 3

Total N (%) Total N (%) P-value

Hg (N = 24) 11 (45.8%) (N = 27) 4 (14.8%) 0.029

Sn (N = 17) 1 (5.9%) (N = 19) 2 (10.5%) 0.999

Au (N = 10) 1 (10.0%) (N = 9) 4 (44.4%) 0.141

Pd (N = 8) 0 (0.0%) (N = 10) 0 (0.0%) -

Cu (N = 18) 1 (5.6%) (N = 25) 0 (0.0%) 0.419

Ni (N = 24) 11(45.8%) (N = 27) 10 (37.0%) 0.578

Co (N = 18) 4 (22.2%) (N = 27) 1 (3.7%) 0.141

Cr (N = 19) 2 (10.5%) (N = 27) 1 (3.7%) 0.561

Mo (N = 11) 0 (0.0%) (N = 8) 0 (0.0%) -

Mn (N = 9) 0 (0.0%) (N = 9) 0 (0.0%) -

Ag (N = 23) 4 (17.4%) (N = 27) 1 (3.7%) 0.167

TiSO4 (N = 18) 0 (0.0%) (N = 13) 0 (0.0%) -

Cd (N = 10) 3 (30.0%) (N = 7) 3 (42.9%) 0.644

Pb (N = 10) 3 (30.0%) (N = 7) 0 (0.0%) 0.228

TiO2 (N = 5) 0 (0.0%) (N = 10) 2 (20.0%) 0.524

Combination* (N = 24) 23 (95.8%) (N = 27) 16 (59.3%) 0.003

P-value of Fisher’s exact test in categorical variables.

* At least one marker has the value� 3.

doi:10.1371/journal.pone.0164786.t003

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 6 / 12

positive response to two or more metals [21]. These results are comparable with those ofhealthy controls in our study.

According to environmental questionnaires, metal pollutants to which our patients hadhypersensitive reactions corresponded to dental filling materials, but also to other environmen-tal burden. For example, mercury, tin, silver and copper are components of dental amalgam,while alloys of nickel, chromium and cobalt are employed to shape crowns and bridges, or tobind dental ceramics. Metals such as aluminium, chromium, cadmium, copper, nickel or lead,which are present in the tobacco, might be the cause of hypersensitivity reactions in both activeand passive smokers [26].

Previous studies have pointed out that there might be an association between hypersensitiv-ity reactions to metals and autoimmune thyroiditis [19] or some other autoimmune diseases[21,27]. Depending on the individual reactivity of a specific patient (determined by a geneticpredisposition), hypersensitivity reactions to metals might–or might not–develop. Accordingto results of HLA typing, patients showing hypersensitivity reactions to metal dental fillingmaterials had significantly increased levels of HLA-B37, B47 and DR4 antigens [28].

Possible pathophysiological mechanism of TS in relation to

hypersensitivity reactions

Development of an acute TS attack is most frequently associated with elevated levels of cate-cholamines [2,5], but the pathological mechanism for the development of cardiac dysfunctionis not fully understood. Development of coronary microvascular spasm is one possibility.Excessively high levels of catecholamines can occur as a result of increased synthesis or reduceddegradation of catecholamines, or the combination of both. During a stress response, there is ageneral increase in the levels of circulating catecholamines. Catechol-O-methyl transferase(COMT) is one of the enzymes involved in the degradation of catecholamines. The methio-nine-homocysteine cycle provides methyl groups for this process catalysed by COMT [29,30].Moreover, methionine-homocysteine cycle is the main endogenous antioxidant system,because it is capable of binding unpaired electrons of free radicals. Cytoplasm, mitochondriaand nuclei of healthy cells and tissues contain predominantly the reduced form of glutathione(GSH), accounting for up to 99% of the total glutathione pool. The reduced form of glutathioneacts as an antioxidant. On the other hand, endoplasmic reticulum contains a large proportion(up to 30%) of glutathione in the oxidised form, the glutathione disulphide (GSSG). Adecreased GSH-to-GSSG ratio is considered indicative of oxidative stress [29]. The methio-nine-homocysteine cycle also has chelation and detoxification functions [29,31]. Metal pollut-ants have a high affinity for thiol groups (–SH) of the reduced form of glutathione (GSH), formcovalent bonds, and thus can be eliminated from the body.

Chronic hypersensitivity reactions are accompanied by inflammatory reactions with theproduction of many cytokines and an increased oxidative stress [32]. A decreased performanceor even the “exhaustion” of the methionine-homocysteine cycle, for example as a consequenceof chronic inflammatory reaction and increased oxidative stress, as indicated above, can lead toa decreased degradation of catecholamines, among others, and therefore to an elevation oftheir plasma levels and an increase in their toxicity. The methionine-homocysteine cycle mightrepresent a key link between metal pollutants, hypersensitivity reactions to metals, detoxifica-tion and antioxidant processes, and catecholamine metabolism; these processes are in somekind of dynamic equilibrium under “resting” conditions (Fig 1). No mechanisms are known toassociate the hypersensitivity reaction with possible coronary microvascular spasm. Instead,vessel reactivity might possibly be influenced by the above-described chronic processes linkedto an increased oxidative stress and chronic inflammation.

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 7 / 12

A disturbance of this equilibrium might apparently manifest as an acute TS attack evenunder less common clinical situations, for example during treatment with allopurinol, statins,or after pacemaker implantation [8–10,17,18,33]. In this context, it must be taken into accountthat many medications–mainly their peroral forms–contain metals as adjuvants (e.g. titaniumdioxide), and that a hypersensitivity reaction can develop either to the active ingredient, or tothe adjuvant.

TS mostly affects postmenopausal women (they are affected about 9 times more often thanmen). Possible role of female sex hormones, especially oestrogens, has therefore been sug-gested. They reduce chronotropic and inotropic effects of catecholamines and are cardioprotec-tive in general [34]. When considering hypersensitive reactivity in the aethiopatogenesis of TS,progesterone is known to have immunosuppressive effects, sometimes is even referred to asnatural immunosuppressive [35,36]. In menopause, decreased female hormone levels mayserve as contributing factors to the development of acute TS attack. Hypersensitivity or autoim-mune reactions can also lead to acute myocarditis, which might imitate TS [37]. MRI examina-tion can be helpful in the differential diagnosis of myocarditis: late gadolinium enhancement(LGE) is typical of myocarditis, but not seen in TS [38]. Subsiding of inflammation in myocar-ditis can lead to an recovery of left ventricular function, similarly to that occurring in TS cases[39].

The possible link between TS development and pathological immune reactivity is docu-mented by the case of one of our patients, who was hospitalised three times for an acute TSattack in a two-year period. A detailed examination newly revealed bronchial asthma with asevere allergic reaction to moulds. After changing residence (and thus removing exposure tothe allergen) and starting asthma treatment, the TS attack did not recur over the next 4 years.

Some studies have described improvements in the condition of patients with autoimmunediseases after the removal of hypersensitivity-provoking materials, such as incompatible metaldental filling materials. This is attributed to the “downregulation” of cellular immunity [40,41].

Limitations

Our study has several limitations. First, TS patients were examined by LTT-MELISA1 4months to 6 years after an acute TS attack, which might seem a large time span. However, weworked on the assumption that patients’ immune reactions to metals do not change signifi-cantly, unless there is a radical change of environmental burden. Such a change was notfound in the questionnaires. Nevertheless, it would be surely interesting to compare levelsmeasured by LTT-MELISA1 in patients in the acute phase with those measured in stablepatients in a stable condition. It would also be interesting to evaluate other biochemicalparameters in the acute phase, such as the levels of catecholamines, cortisol, corticotropin-releasing hormone, or oxidative stress markers. Second, our study was monocentric, andinvolved a relatively small number of evaluated persons both in the group of TS patients andin the control group of healthy individuals. Despite this limitation, it is obvious that hyper-sensitivity reactions to metals are very common in TS patients. Third, in both groups (TCMpatients and controls), hypersensitivity reactions were only evaluated for metals selected bythe questionnaire of environmental burden. Hypersensitivity reactions to metals that had notbeen evaluated cannot be ruled out in either of the two groups. Fourth, it is widely knownthat hypersensitivity reactions are attenuated by glucocorticoids. In our cohort of TSpatients, five patients (20%) were on glucocorticoid therapy: four patients used glucocorti-coid spray to treat COPD or bronchial asthma, one patient had systemic glucocorticoid ther-apy to treat rheumatoid arthritis. Although immune reactions were suppressed in thesepatients, hypersensitivity reactions to 1 or 2 metals (SI� 3) were detected in all of them.

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 8 / 12

Fig 1. Suggested hypothesis of pathological immune mechanisms in the Takotsubo syndrome. TS is associated with increased levels of

catecholamines in the acute phase of this condition. Hypersensitivity reactions to environmental burden with metals, which are mediated by antigen/

hapten-specific T-lymphocytes, were proved in 96% of our patients with Takotsubo syndrome. Chronic or acute hypersensitivity reactions are

accompanied by an inflammatory reaction with the production of many cytokines and free radicals (i.e., an increased oxidative stress). The

methionin-homocysteine cycle is one of the main endogenous antioxidant systems. At the same time, the methionine-homocysteine cycle provides

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 9 / 12

Conclusion

TS is a rare condition with unclear aetiology. Our work shows that conditions with pathologicalimmune reactivity occur frequently in TS patients and our results support the hypothesis of apossible association between TS and hypersensitivity to metal pollutants (mercury in particu-lar) evaluated by LTT-MELISA1. The suggested pathophysiological mechanisms need to befurther evaluated and verified on a large cohort of patients.

Our work also suggests that apart from the triggering stress factor, potential existence ofother serious conditions should also be considered when taking medical history of TS patients.

Supporting Information

S1 File. Questionnaire of environmental burden.(DOCX)

Acknowledgments

We thank the staff of Laboratoire MGD in Switzerland for their excellent cooperation in theLTT-MELISA1 testing.

Author Contributions

Conceptualization: JM VS P. Kubena PL P. Kala RM JS JP.

Data curation: JJ KB JP.

Formal analysis: JM JJ KB JP.

Funding acquisition: JM PN JS JP.

Investigation: JM P. Kubena PL LD AZ MP JP.

Methodology: JM VS JJ PN LD AZ RM JP.

Project administration: JM AZ LD.

Resources: JM PN LD AZ RM JS JP.

Supervision: JP.

Validation: P. Kala JS.

Visualization: JM JJ KB RM.

Writing – original draft: JM VS PN MP JS JP.

Writing – review& editing: JM VS P. Kubena JJ PN PL LD AZ MP KB P. Kala RM JS JP.

methyl groups for the degradation of catecholamines, a process catalysed by catechol-O-methyltransferase (COMT). An “exhaustion” of the

methionine-homocysteine cycle as a result of chronic oxidative stress can lead to a decreased degradation of catecholamines; in a subsequent

stressful situation, which is generally characterised by an increased production of catecholamines, excessively high levels of catecholamines can

occur, such as those typically found in TS patients. TS develops most frequently in postmenopausal women, who have decreased levels of sex

hormones, particularly oestrogens. In general, oestrogens have a cardioprotective effect (by reducing the chronotropic and ionotropic effects of

catecholamines); additionally, when taking into account the potential hypersensitivity, oestrogens also have anti-inflammatory effects. On top of that,

progesterone–of which levels also decrease with age–has immunosuppressive effects.

doi:10.1371/journal.pone.0164786.g001

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 10 / 12

References1. Sato H, TH, Dote K UT. Tako-tsubo-like left ventricular dysfunction due to multivessel coronary spasm.

Clinical aspect of myocardial injury: From ischemia to heart failure. Tokyo: Kagakuhyoronsha Publish-

ing Co; pp. 56–64.

2. Akashi YJ, Goldstein DS, Barbaro G, Ueyama T. Takotsubo Cardiomyopathy A New Form of Acute,

Reversible Heart Failure. Circulation. 2008; 118: 2754–2762. doi: 10.1161/CIRCULATIONAHA.108.

767012 PMID: 19106400

3. Bybee KA, Kara T, Prasad A, Lerman A, Barsness GW, Wright RS, et al. Systematic review: transient

left ventricular apical ballooning: a syndrome that mimics ST-segment elevation myocardial infarction.

Ann Intern Med. 2004; 141: 858–865. PMID: 15583228

4. Templin C, Ghadri JR, Diekmann J, Napp LC, Bataiosu DR, Jaguszewski M, et al. Clinical Features

and Outcomes of Takotsubo (Stress) Cardiomyopathy. N Engl J Med. 2015; 373: 929–938. doi: 10.

1056/NEJMoa1406761 PMID: 26332547

5. Akashi YJ, Nef HM, Lyon AR. Epidemiology and pathophysiology of Takotsubo syndrome. Nat Rev

Cardiol. 2015; 12: 387–397. doi: 10.1038/nrcardio.2015.39 PMID: 25855605

6. Lyon AR, Bossone E, Schneider B, Sechtem U, Citro R, Underwood SR, et al. Current state of knowl-

edge on Takotsubo syndrome: a Position Statement from the Taskforce on Takotsubo Syndrome of

the Heart Failure Association of the European Society of Cardiology. Eur J Heart Fail. 2016; 18: 8–27.

doi: 10.1002/ejhf.424 PMID: 26548803

7. Pelliccia F, Parodi G, Greco C, Antoniucci D, Brenner R, Bossone E, et al. Comorbidities frequency in

Takotsubo syndrome: an international collaborative systematic review including 1109 patients. Am J

Med. 2015; 128: 654.e11–19. doi: 10.1016/j.amjmed.2015.01.016 PMID: 25660245

8. Knott K, Starling N, Rasheed S, Foran J, Cafferkey C, Rosen S, et al. A case of Takotsubo syndrome

following 5-fluorouracil chemotherapy. Int J Cardiol. 2014; 177: e65–67. doi: 10.1016/j.ijcard.2014.09.

154 PMID: 25449494

9. Koci F, Eltibi R, Hadley M, Kumar D. Necrotic bowel induces takotsubo-like myocardial injury. Tex

Heart Inst J Tex Heart Inst St Lukes Episcop Hosp Tex Child Hosp. 2014; 41: 638–640. doi: 10.14503/

THIJ-13-3500 PMID: 25593531

10. Han L, Cui L, Yin L, Zhou C. Takotsubo cardiomyopathy induced by epileptic seizure. Int J Cardiol.

2014; 177: e145–146. doi: 10.1016/j.ijcard.2014.09.049 PMID: 25294179

11. AndòG, Trio O, de Gregorio C. Transient left ventricular dysfunction in patients with neurovascular

events. Acute Card Care. 2010; 12: 70–74. doi: 10.3109/17482941003732758 PMID: 20384473

12. Trio O, de Gregorio C, AndòG. Myocardial dysfunction after subarachnoid haemorrhage and tako-

tsubo cardiomyopathy: a differential diagnosis? Ther Adv Cardiovasc Dis. 2010; 4: 105–107. doi: 10.

1177/1753944709356013 PMID: 20124311

13. Kuběna P, Bohata S, Maňousek J, Spinar J, Pařenica J. [Takotsubo cardiomyopathy, clinical experi-

ence with the disease and one-year prognosis of patients]. Vnitrnı Lekarstvı. 2015; 61: 619–625.

PMID: 26375687

14. Amariles P. A comprehensive literature search: drugs as possible triggers of Takotsubo cardiomyopa-

thy. Curr Clin Pharmacol. 2011; 6: 1–11. PMID: 21235467

15. Bernards J, Van Kolen K, Govaerts E, Somville FJ, von Stritzky M. Transient left ventricular apical bal-

looning syndrome in a patient with allopurinol induced skin rash: a case report. Int J Cardiol. 2014; 177:

e53–55. doi: 10.1016/j.ijcard.2014.09.130 PMID: 25305677

16. Basselin C, Fontanges T, Descotes J, Chevalier P, Bui-Xuan B, Feinard G, et al. 5-Fluorouracil-

induced Tako-Tsubo-like syndrome. Pharmacotherapy. 2011; 31: 226. doi: 10.1592/phco.31.2.226

PMID: 21275498

17. Coli S, Pigazzani F, Gaibazzi N. Midventricular Takotsubo cardiomyopathy after oxaliplatin infusion:

an unreported side effect. J Cardiovasc Med Hagerstown Md. 2015; 16: 646–649. doi: 10.2459/JCM.

0000000000000204 PMID: 25333375

18. Postema PG, Wiersma JJ, van der Bilt I a. C Dekkers P, van Bergen PFMM. Takotsubo cardiomyopa-

thy shortly following pacemaker implantation-case report and review of the literature. Neth Heart J Mon

J Neth Soc Cardiol Neth Heart Found. 2014; 22: 456–459. doi: 10.1007/s12471-012-0320-8 PMID:

23055052

19. Hybenova M, Hrda P, Prochazkova J, Stejskal V, Sterzl I. The role of environmental factors in autoim-

mune thyroiditis. Neuro Endocrinol Lett. 2010; 31: 283–289. PMID: 20588228

20. Saint-Mezard P, Berard F, Dubois B, Kaiserlian D, Nicolas J-F. The role of CD4+ and CD8+ T cells in

contact hypersensitivity and allergic contact dermatitis. Eur J Dermatol EJD. 2004; 14: 131–138.

PMID: 15246935

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 11 / 12

21. Stejskal V, Reynolds T, Bjørklund G. Increased frequency of delayed type hypersensitivity to metals in

patients with connective tissue disease. J Trace Elem Med Biol Organ Soc Miner Trace Elem GMS.

2015; 31: 230–236. doi: 10.1016/j.jtemb.2015.01.001 PMID: 25636536

22. Stejskal VD, Cederbrant K, Lindvall A, Forsbeck M. MELISA-an in vitro tool for the study of metal

allergy. Toxicol Vitro Int J Publ Assoc BIBRA. 1994; 8: 991–1000.

23. Stejskal VD, Forsbeck M, Cederbrant KE, Asteman O. Mercury-specific lymphocytes: an indication of

mercury allergy in man. J Clin Immunol. 1996; 16: 31–40. PMID: 8926283

24. Thyssen JP, Menne T. Metal allergy—a review on exposures, penetration, genetics, prevalence, and

clinical implications. Chem Res Toxicol. 2010; 23: 309–318. doi: 10.1021/tx9002726 PMID: 19831422

25. Smith-Sivertsen T, Dotterud LK, Lund E. Nickel allergy and its relationship with local nickel pollution,

ear piercing, and atopic dermatitis: a population-based study from Norway. J Am Acad Dermatol.

1999; 40: 726–735. PMID: 10321601

26. Bernhard D, Rossmann A, Wick G. Metals in cigarette smoke. IUBMB Life. 2005; 57: 805–809. doi: 10.

1080/15216540500459667 PMID: 16393783

27. Stejskal J, Stejskal VD. The role of metals in autoimmunity and the link to neuroendocrinology. Neuro

Endocrinol Lett. 1999; 20: 351–364. PMID: 11458198

28. Prochazkova J, Bartova J, Ivaskova E, Kupkova L, Sterzl I, Stejskal VD. HLA-association in patients

with intolerance to mercury and other metals in dental materials. Dis Markers. 2000; 16: 135–138. doi:

10.1155/2000/205757 PMID: 11381194

29. Lushchak VI. Glutathione homeostasis and functions: potential targets for medical interventions. J

Amino Acids. 2012; 2012: 736837. doi: 10.1155/2012/736837 PMID: 22500213

30. James SJ, Cutler P, Melnyk S, Jernigan S, Janak L, Gaylor DW, et al. Metabolic biomarkers of

increased oxidative stress and impaired methylation capacity in children with autism. Am J Clin Nutr.

2004; 80: 1611–1617. PMID: 15585776

31. Mutter J. Is dental amalgam safe for humans? The opinion of the scientific committee of the European

Commission. J Occup Med Toxicol Lond Engl. 2011; 6: 2. doi: 10.1186/1745-6673-6-2 PMID:

21232090

32. Hwang S-A, Actor JK. Hypersensitivity: T Lymphocyte Mediated (Type IV). eLS. John Wiley & Sons,

Ltd; 2001. Available: http://onlinelibrary.wiley.com/doi/10.1002/9780470015902.a0001139.pub3/

abstract

33. Kamada T, Hayashi M, Yokoi H, Fujiwara W, Yoshikawa D, Mukaide D, et al. Takotsubo cardiomyopa-

thy with involvement of delayed-onset rhabdomyolysis and acute kidney injury after rosuvastatin treat-

ment. Intern Med Tokyo Jpn. 2015; 54: 31–35. doi: 10.2169/internalmedicine.54.3239 PMID:

25742890

34. Kneale BJ, Chowienczyk PJ, Brett SE, Coltart DJ, Ritter JM. Gender differences in sensitivity to adren-

ergic agonists of forearm resistance vasculature. J Am Coll Cardiol. 2000; 36: 1233–1238. PMID:

11028476

35. Szekeres-Bartho J, Hadnagy J, Pacsa AS. The suppressive effect of progesterone on lymphocyte

cytotoxicity: unique progesterone sensitivity of pregnancy lymphocytes. J Reprod Immunol. 1985; 7:

121–128. PMID: 3981485

36. Siiteri PK, Febres F, Clemens LE, Chang RJ, Gondos B, Stites D. Progesterone and maintenance of

pregnancy: is progesterone nature’s immunosuppressant? Ann N Y Acad Sci. 1977; 286: 384–397.

PMID: 152594

37. Caforio ALP, Tona F, Vinci A, Calabrese F, Ramondo A, Cacciavillani L, et al. Acute biopsy-proven

lymphocytic myocarditis mimicking Takotsubo cardiomyopathy. Eur J Heart Fail. 2009; 11: 428–431.

doi: 10.1093/eurjhf/hfp008 PMID: 19193625

38. Eitel I, Lucke C, Grothoff M, Sareban M, Schuler G, Thiele H, et al. Inflammation in takotsubo cardio-

myopathy: insights from cardiovascular magnetic resonance imaging. Eur Radiol. 2010; 20: 422–431.

doi: 10.1007/s00330-009-1549-5 PMID: 19705125

39. Krejci J, Hude P, Poloczkova H, Zampachova V, Stepanova R, Freiberger T, et al. Correlations of the

changes in bioptic findings with echocardiographic, clinical and laboratory parameters in patients with

inflammatory cardiomyopathy. Heart Vessels. 2014; doi: 10.1007/s00380-014-0618-0 PMID:

25539624

40. Yaqob A, Danersund A, Stejskal VD, Lindvall A, Hudecek R, Lindh U. Metal-specific lymphocyte reac-

tivity is downregulated after dental metal replacement. Neuro Endocrinol Lett. 2006; 27: 189–197.

PMID: 16648791

41. Prochazkova J, Sterzl I, Kucerova H, Bartova J, Stejskal VDM. The beneficial effect of amalgam

replacement on health in patients with autoimmunity. Neuro Endocrinol Lett. 2004; 25: 211–218.

PMID: 15349088

Takotsubo Syndrome and Delayed-Type Hypersensitivity

PLOS ONE | DOI:10.1371/journal.pone.0164786 November 8, 2016 12 / 12